The Prevalence of Lymphatic Filariasis in Elementary School Children Living in Endemic Areas: A Baseline Survey Prior to Mass Drug Administration in Pekalongan District-Indonesia.

Background: WHO initiated lymphatic filariasis (LF) elimination globally. Pekalongan District, as LF endemic area, started a program of mass drug administration (MDA) to combat LF in 2015. This study aimed to determine prevalence of Wuchereria bancrofti infection prior to the MDA. Methods: LF infection was detected by the existence of circulating filarial antigen (CFA) W. bancrofti using immunochromatographic card test (ICT). The study population consisted of 1404 elementary school (ES) students living in Pekalongan District. Overall, 1033 were selected as study subjects. Prevalence survey was also conducted on 436 general population in areas where infected students were found. Results: The subjects ranged from 7–17 yr old (mean 9.85±1.296) and equally distributed between both sexes. Prevalence of W. bancrofti infection was 1.98% in children. Infection was mostly found in older students (12 yr old), male, in 6th grade, but did not differ significantly (P=0.129, 0.376, and 0.212 respectively). On the other hand, distribution of infection was significantly different by school (P=0.009) and sub-district (P=0000). Most of children with LF infection were found in Tirto Sub District. In general population, the prevalence of W. bancrofti infection in Tirto was 4.4%. Proportion of infection in males (12.2%) was greater than females (3.8%), with 78.9% of positive cases were in adult over 20 yr old. Conclusion: Cases of W. bancrofti infection exist in Pekalongan District, both in children and adults. Implementation of MDA must be carefully monitored in order to achieve elimination target.


Introduction
Lymphatic filariasis (LF) is a chronic infectious disease caused by filarial worms Wuchereria bancrofti, Brugia malayi, and B. timori (1,2). The disease is a main community health problem, which mostly affects susceptible people of all ages and sexes (3,4) especially in tropical and subtropical countries (5). About 120 million people in 58 countries are infected globally with an estimated 1.23 billion at risk of infection (6). The disease usually had a very low attention (neglected) in the countries where it is prevalent (7). The manifestations of the disease are noticeably disfiguring (2,3,8), including lymphedema of the limbs, male genitalia (hydrocele), or swollen breast (2). WHO launched Global Program to Eliminate Lymphatic Filariasis (GPELF) in 2000. The GPELF targeted the elimination of LF as public health problem by 2020 through mass drug administration (MDA) (9). From 2000 to 2013, WHO had delivered more than 5 billion doses of anti-filarial drugs to almost 984 million at-risk individuals worldwide (6). In 2015, Indonesia planned to carry out MDA in 106 LF endemic districts (10). Pekalongan District is an endemic LF area with 62 cases chronic LF, which potentially will continue to increase and spread if not promptly treated. "Effective monitoring and evaluation are necessary to achieve the goals of LF elimination" (11). After MDA, LF programs must be able to assess whether the prevalence of infection decrease to a level at which transmission is no longer likely to be sustainable (11). Pekalongan District had conducted partial treatment of LF only in its endemic areas (five sub-districts) since 2002. However, data from the District Health Office (DHO) of Pekalongan showed mf rate after the partial MDA (2003-2007) remained high, ranged from 1.15% to 3.90%. Pekalongan District re-implemented MDA in all sub-districts by 2015. Prior to the MDA, there was no recent data on LF prevalence. Therefore, a baseline survey should be conducted to determine the existence of new infection. LF infection in children is a marker for recent exposure to the parasite (12). In this study, we conducted a survey prior to the MDA. The study aimed to determine prevalence of W. bancrofti infection among elementary school students in endemic sub-districts. The prevalence data will be used as a baseline data because of LF elimination programs will be applied to the whole district.

Ethics Statement
Ethical approval was obtained from the Committee of Public Health Research Ethics, Diponegoro University (107/EC/FK/2015). Informed consent was acquired from students, in addition to written consent from their parents. Written informed consent was obtained from teachers.

Study design
This was a descriptive study designed as baseline data to measure prevalence of W. bancrofti prior to MDA. Selection of the study site was based on endemicity of LF. Endemicity data was obtained from DHO of Pekalongan. The study was carried out in five LF endemic sub-districts, namely: Buaran, Kedungwuni, Tirto, Wiradesa, and Wonokerto (mf rate in 2007 were 3.9%, 1.27%, 1.15%, 1.40%, and 1.23% respectively). There were a total of 128 elementary schools (ES) in those sub-districts, 28 in Wiradesa, 24 in Wonokerto, 40 in Kedungwuni, 20 in Tirto, and 16 in Buaran. Fifteen ES from those areas were randomly selected. The protocol for this study was reviewed and approved by the Health Research Ethical Review Committees of the Public Health Faculty, Diponegoro University, Indonesia. The survey was conducted in Sep 2015, a month before the first round of MDA was implemented.

Subject
Inclusion criteria consisted of students from selected ES, in 3 rd to 6 th grade, and willing to join the study. Study population consisted of 1404 ES students (729 male and 675 female) living in five LF endemic sub-districts in Pekalongan. Of 1404 students, 1033 (98.9%) agreed to undergo CFA examination, as outlined in Fig. 1. Prevalence survey was also conducted in general population of Tirto Sub District, where LF infected students were highest.

Blood drawing and circulating filarial antigen detection
Circulating filarial antigen (CFA) detection used immunochromatographic test (ICT) from Alere Scarborough Inc., Scarborough -ME USA. A volume of 100 μL blood was drawn by finger prick using microtainer BD blue. The blood was collected in a calibrated capillary tube of each individual. Blood was then added to the white portion of the sample pad of the card according to the manufacturer protocol. Once the card was closed, the timer was started. The result of the test was read exactly 10 min after closing the card. Pink control (C) and test (T) lines were visible for all valid positive tests, whereas only the control pink line appeared for negative tests.

Mapping
Mapping of LF used GIS software to produce graphic displays of geographical information. The map showed distribution of LF infection and chronic cases in Pekalongan District. Map of Pekalongan District was obtained from Dinas Tata Ruang Kabupaten Pekalongan. Coordinate of LF infection and chronic cases were obtained from GPS.

Analysis
Data were presented as proportion, mean ± deviation standard, minimum, and maximum. Statistical analysis used Chi-Square to test the difference between positive and negative cases based on sex, grade, school, and sub-district. Mean of age was compared by independent t-test.

Results
Overall, 1033 students were selected from 15 ES. Among 1033 subjects examined, there were 275 (26.6%) invalid result due to rapid blood clot. The result showed 758 valid results, and 15 subjects were infected by W. bancrofti, as outlined in Fig. 1. Prevalence of W. bancrofti antigenemia in students was therefore 1.98%.Characteristics of study subjects can be seen in Table 1.   Fig. 2 shows geographical distribution of chronic and active cases of LF. Most chronic cases were found in Tirto Sub District.
Chronic cases also sporadically spread in other sub districts (Wiradesa, Buaran, Kedungwuni, Wonopringgo, Kajen, Kesesi, Doro, Petungkriono, and Paninggaran).   (13). Tirto is qualified to be a sentinel site when MDA evaluation takes place. Once a sentinel site is selected, it should continue to serve as the sentinel site throughout the program. "Blood surveys at sentinel sites are used to establish the baseline infection level and to monitor the impact of MDA on infection prevalence periodically" (13). Pekalongan District has implemented partial MDA in five endemic sub districts, started in 2002 and ended in 2007 (14). Yet this study found prevalence of LF remains higher than the level of transmission threshold (15,16). According to WHO, after five years of MDA, the expected prevalence of CFA should not exceed 2% (16). With an overall prevalence of 1.98% in children and 4.4% in population, LF remains a public health problem in Pekalongan, especially in Tirto Sub District. Although the prevalence is lower than that of earlier observations (2014) in Indonesia (4.7%) (10), infection in children should receive attention. LF in children is a marker for relatively recent exposure to the parasite (12) and is a sensitive indicator of LF endemicity (17). This study supports the need for targeting children in LF elimination campaign. "LF prevalence in Indonesia varied from 0.5 to 27.6%. The high rates of LF were found in Maluku, Papua, West Papua, East Nusa Tenggara, and North Maluku" (18). All people at risk are involved in this program (16). The efficacy of six annual rounds of MDA was studied in Alor Island, eastern part of Indonesia. Microfilaria rates in Alor decreased significantly after MDA intervention, from 26% to 0.17%. MDA may be recommended for other parts of Indonesia (19). However, the challenge of MDA was mostly related to the infrastructure of MDA implementation (20) and the compliance with drug administration (21). Therefore, LF elimination in remote area in Indonesia is a major challenge (22). This result showed the prevalence of LF in children increased with age. The similar type of prevalence was also reported from other endemic areas (23)(24)(25). Increasing of LF prevalence according to the age in an endemic area correlates with the duration of exposure to the infection (23,26). In this study, the existence of concurrent prevalence of LF both in children and adults was observed in Tirto Sub Districts. Transmission had occurred (27) in the area. Knowledge of LF prevalence in children is very important for understanding the future status of the disease. In this case, the global elimination program decided to protect children from LF (28,29), because children who are close to adults are more exposed to infection (4). This result will help to develop public health strategies for treatment of LF infection in children and to reduce the future disease burden in the adult population. This study confirmed previous epidemiological studies that proportion of LF was higher in male than female (4,23,25). Higher prevalence in male usually due to the possibility to be exposed to mosquitoes (4). If left untreated, they may experience lymphatic damages and develop hydrocele by around 10-15 yr old, because the parasites seem to prefer the lymphatic of scrotum (30). This study also found LF cases between families, with all family members were infected. The clustering of LF cases possibly due to genetic (31)(32)(33)(34)(35)(36) or environmental (34,37) factors. A study in Mauke, Pacific Island revealed a strong association of genetic and LF in population (36). Several other studies in LF endemic areas also showed the infection and microfilariae burden tend to cluster in families, which was mostly due to genetic factor (31)(32)(33)(34)(35)(36).
As control effort is implemented, maps of the progress in control can help highlight success and indicate where further effort is required. Mapping of LF was conducted to show the distribution of active LF infection and chronic cases in Pekalongan District (Fig. 2). Both chronic cases in older population and infection in children were mostly found in Tirto Sub District. Tirto is lowland, which located 4 m above sea level. There are two coastal areas in the sub district. Several villages experience tidal inundation and are continuously affected by stagnant water or flood. The existence of stagnant water may serve as breeding places of LF vector Culex quinquefascitus (38

Cases of W. bancrofti infection exist in Pekalongan
District, both in children (1.98%) and adults (4.4%). LF infection in children is a marker for relatively recent exposure to the parasite and is a sensitive indicator of LF endemicity. Therefore, this result will help to develop public health strategies for treatment of LF infection in children and to reduce the future disease burden in the adult population. Based on geographical distribution, LF infection was found clustering in Tirto Sub-district, located on the northern coast of Central Java. The result supported the potential of Tirto Sub District to serve as sentinel site for Transmission Assessment Survey (TAS). This study may serve as baseline data prior MDA program. Monitoring and evaluation of the program before second round of MDA should be done to measure the prevalence and effect of MDA.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

Funding
This study was financially supported by the Directorate General of the Higher Education, Ministry of Education and Culture and the Research Institute of Diponegoro University (grant number 140-17/UN7.5.1/PG/2015). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.